frameshift after ligation - point-mutation in restriction site (Nov/03/2004 )

We tried to clone a 700 bp fragment, amplified with PCR, in our 5.4 kb vector. One main problem is, that both are digested with EcoRI and MscI (blunt end). So therefore, we dephosphorylated the vector by using CIAP, we ligated the insert in a 1 : 3 molar ratio with some more units of ligase, with PEG and also overnight at 16 °C and transformed it in E. coli Nova Blue. We picked some clones for doing a colony PCR and then we let the positive ones grow over night. Well, now the big main problem is, that we are not able, to cut out the insert of our positive clones, which have the insert for sure...! The blunt end restriction site MscI is no longer working. And so I am asking myself, is it possible that the enzyme MscI might cut a kind of sticky end with 1 bp overhang (however), so that the insert might be ligated with a single-strand break and the bacteria fill the missing base?!? Might the sequence TGGCCA be susceptible to mutations?I would be glad for further information...

-Janina-

This is most likely a result of nibbling of the Msc I-cut vector or insert by exonuclease contaminants in the restriction enzymes or the CIAP. It is probable that some clones escaped this nibbling, so check more colonies.

-tfitzwater-

Thanks for responding that quick.

But the point is that we have one base pair too much, so that the possibility of a exonuclease contaminant doesn't fit.

-Janina-

So you've sequenced it, since you know you've got an extra base?!

You don't have to use CIAP when you are using two different enzymes... alternatively, you could put the pcr product into TOPO and then cut out from there. Then you'll know you've got the right ends...

Have you tried to cut with EcoRI only and thus seen a shift in weight?